Enzyme activity measurements are clinical biomarkers for organ or muscle function. The detection principles disclosed in this document can be applied to hydrolases: enzymes that catalyze hydrolysis that results in the cleavage of an enzyme-specific side-group from the rest of the substrate. Examples include alkaline phosphatase (ALP), cholinesterase, and, esterase. ALP measurements are discussed in detail to illustrate the general detection method which may be applied for any of the mentioned enzymes. ALP is an enzyme included in standard liver panel assays and is a marker of cholestatic hepatoxicity. A higher than normal level of ALP may indicate that the subject of the test has liver disease, or cancer of the liver or bones.
Existing ALP assays measure enzyme activity using a chromogenic substrate consisting of 4-nitrophenyl phosphate. The chromogenic substrate, placed in an alkaline environment, changes to a yellow color in the presence of ALP. The color change is quantified by measuring the absorption spectrum using a spectrophotometer. Enzyme activity in chromogenic assays depends upon the reagent buffer's ability to revitalize the enzyme, pH, and the preservation of the blood specimen. Additional disadvantages of chromogenic assays are the production of precipitates that may interfere with enzyme activity thereby reducing sensitivity.
The chromogenic assays may be further complicated by interference caused by quenching from hemoglobin in red blood cells. This level of interference may reduce the sensitivity of chromogenic assays and possibly mask the presence of some low level enzymes.
Thus, a need still remains for an enzyme detection system with caged substrates that may improve the efficiency of whole blood assays. In view of the aging world population, it is increasingly critical that answers be found to these problems. With extended life expectancy and the development of many new drugs to support it, an efficient and cost effective enzyme detection system is a primary concern. Additionally, the need to save costs, improve efficiencies and performance, and meet competitive pressures, adds an even greater urgency to the critical necessity for finding answers to these problems.
Solutions to these problems have been long sought but prior developments have not taught or suggested any solutions and, thus, solutions to these problems have long eluded those skilled in the art.